Recombinant Cells and Organisms Having Persistent Nonstandard Amino Acid Dependence and Methods of Making Them

ABSTRACT

Novel recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making novel recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

RELATED APPLICATION

This application is a continuation application which claims priority toU.S. patent application Ser. No. 15/025,406, filed on Mar. 28, 2016,which is a National Stage Application under 35 U.S.C. 371 of co-pendingPCT application PCT/US2014/057573 designating the United States andfiled Sep. 26, 2014; which claims the benefit of provisional application61/883,413 and filed Sep. 27, 2013 each of which are hereby incorporatedby reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under EEC-0540879awarded by National Science Foundation and under DE-FG02-02ER63445awarded by U.S. Department of Energy and under N66001-12-C-4040 awardedby U.S. Department of Defense Space and Naval Warfare Systems CommandThe government has certain rights in the invention.

FIELD

The present invention relates in general to genetically modified cellsand/or organisms.

BACKGROUND

Genetically modified organisms are increasingly used to produce humanconsumables such as fuels (e.g., ginkgo, LS9, Solazyme, Chromatin),commodity chemicals (e.g., Genencor, Genomatica, Verdezyne), andtherapeutics (e.g., Ambrx, Amyris). They are also used in agriculture(e.g., Golden Rice, Roundup Ready crops, Frostban), bioremediation(e.g., oil spills), and healthcare (e.g., Crone's disease and oralinflammation). Although bio-containment strategies, such as engineeredauxotrophy, induced lethality and gene flow prevention have beendeveloped, selective pressure from leaky containment mechanisms can leadto bio-containment escape.

Genetically modified organisms provide unique opportunities to broadenthe functional repertoire accessible to biotechnology. By reassigningthe genetic code, organisms can produce proteins incorporatingnonstandard amino acids (NSAAs) with properties not found in nature,resist viral infection due to mistranslated viral transcripts, andmaintain genetic isolation from naturally coded organisms in theirsurroundings (F. J. Isaacs et al. (2011) Precise Manipulation ofChromosomes in Vivo Enables Genome-Wide Codon Replacement. Science333:348). However, such organisms require unprecedented safetymechanisms to ensure their containment.

Current best practices confer dependence on supplemented metaboliteslike diaminopimelic acid (DAP) (R. Curtiss (1978) Biological containmentand cloning vector transmissibility. Journal of Infectious Diseases137:668); J. Santander, W. Xin, Z. Yang, R. Curtiss (2010) TheAspartate-Semialdehyde Dehydrogenase of Edwardsiella ictaluri and ItsUse as Balanced-Lethal System in Fish Vaccinology. PLoS One 5:e15944),an essential component of the cell wall, for survival. Since metaboliteslike DAP are natural products found in the environment, this strategy isinsufficient to ensure containment of genetically modified organisms.Mutations that suppress NSAA dependence, acting either on the recodedgenes or, more likely, on extent tRNAs (G. Eggertsson, D. Söll (1988)Transfer ribonucleic acid-mediated suppression of termination codons inEscherichia coli. Microbiological Reviews 52:354), will compromise anysafety features that rely on genomic recoding.

Auxotrophies for natural metabolites like DAP or Thy are known in theart (R. Curtiss, Supra; J. Santander, Supra), but they lack sufficientsafeguards. This is due to the fact that the metabolites can be found innature, safety tests often focused only on the mammalian gut, andconjugation tests often considered only conjugation out from agenetically modified organism rather than into it, and did not addresstransduction by viruses and or phages.

Kill switches controlling genetically modified organism survival areknown in the art (M. C. Ronchel, J. L. Ramos (2001) Dual system toreinforce biological containment of recombinant bacteria designed forrhizoremediation. Applied and Environmental Microbiology 67:2649), butthese too lack sufficient safeguards. These genetically modifiedorganisms have the potential for escape via failure of the killingmechanism and/or via broken circuitry (e.g., inactivation of repressors,activators, or other expression regulation mechanisms involved in thekill switch, loss of expression of a killing mechanism, constitutiveactivity of a survival mechanism and the like).

Methods of placing NSAAs at surface positions or near the 5′ translationstart site are also known, although these methods are also lacking.These methods incorporate natural amino acids at surface or terminalpositions and are far less likely to disrupt folding and function,leaving clear routes to natural suppression (i.e., a natural tRNAmutates so that is can incorporate a natural amino acid at the desiredposition of the NSAA). Although escape can potentially be mitigated byplacing NSAAs at very many surface and terminal positions, thiscomplicates the approach relative to a much fewer number of high-impactcore mutations and doesn't protect against suppressor mutations in whicha natural amino acid is translated at the reassigned codon.

Horizontal and vertical gene transfer methods are also known.Agriculture and bioremediation use genetically modified organisms in theenvironment. However, methods are needed to prevent escape ofherbicide/antibiotic resistant strains and to prevent cross-pollinationand/or DNA transfer between genetically modified organisms and organiccrops or other natural organisms.

Partial methods for reassigning the genetic code in order to efficientlyincorporate NSAAs during translation are known (T. Mukai et al. (2010)Codon reassignment in the Escherichia coli genetic code. Nucleic AcidsRes. 38:8188; D. Johnson et al., Rf1 knockout allows ribosomalincorporation of unnatural amino acids at multiple sites. Nat Chem Biol.7:779), but these methods are lacking. For example, partial recodingmethods are not scalable/generalizable to other codons, because allgenes would have to be duplicated using the target codon. Additionally,UAG function cannot be abolished because these methods do not remove allinstances of UAG throughout the genome. Accordingly, such methods onlyswap codon function. Furthermore, these methods result in a strongselection pressure for natural suppressors, which leads to an unstablegenetic code.

The engineering of organisms that can only grow safely in well-defined,restrictive environments has been a long-standing goal that dates backto the Asilomar Conference from 1975 and has yet to be achieved.Accordingly, recombinant cells, recombinant organisms (and methods ofmaking them) that avoid unintended survival are needed.

SUMMARY

To prevent unintended survival of genetically modified cells and/ororganisms, additional safeguards need to be implemented. For example,genetically modified organisms should contain modifications that cannotbe escaped by complementation and cannot revert through natural mutationmechanisms. Genetic modifications should be sufficiently inexpensive toallow use in a large bioreactor for production of commodity chemicals.Genetic modifications should not adversely impact the stability,metabolic load, growth rate, or gene expression of the organism. Geneticmodifications should extrapolate to industrially-relevant organisms.

Synthetic auxotrophy, whereby persistence of NSAA dependence ismaintained in genetically modified organisms, is a promising approachfor achieving these additional safeguards (FIG. 2). In order to ensurepersistence of NSAA dependence, two complementary strategies arepresented herein. A positive selection approach is provided hereinwhereby essential nucleic acid sequences (e.g., genes) encode anessential polypeptide sequence relying on the presence of one or moreNSAAs in the polypeptide sequence to ensure translation, folding and/orproper function of the polypeptide (FIG. 1). A negative selectionapproach is provided herein whereby one or more toxins, toxic products,or polypeptides that produce or confer susceptibility to toxins or toxicproducts are misfolded and/or functionally impaired when an NSAA ispresent at a particular position of the one or more toxins, toxicproducts, or polypeptides that produce or confer susceptibility totoxins or toxic products. Each of these approaches may optionally becombined. These approaches, alone or in combination, provide anunprecedented safety mechanism that will advance humankind's ability toprevent unintended survival of genetically modified organisms.

Accordingly, in certain exemplary embodiments, a recombinant cellincluding an essential nucleic acid sequence (e.g., one, two or moreessential nucleic acid sequences) encoding an essential polypeptide(e.g., one, two or more essential polypeptides) having a nonstandardamino acid substitution at a particular position is provided. Theabsence of the nonstandard amino acid substitution at the particularposition disrupts one or any combination of translation, folding andfunction of the essential polypeptide, and the cell dies or experiencesreduced or no proliferation if one or more of translation, folding orfunction of the essential polypeptide is disrupted. In certain aspects,one or more standard amino acid substitutions are present in theessential polypeptide to accommodate the nonstandard amino acidsubstitution and maintain one or both of proper folding and properfunction of the essential polypeptide. In other aspects, the essentialpolypeptide has been computationally (e.g., with software such asRosetta) or empirically redesigned (e.g. by random mutagenesis andselection, by targeted mutagenesis and selection and/or computationaldesign) so that the essential polypeptide can no longer fold properly,function properly or both, when the essential polypeptide expresses astandard amino acid at the particular position. In certain aspects, theessential nucleic acid sequence is a gene or encodes an essentialpolypeptide that is essential for survival, growth, or proliferation. Inother aspects, the essential polypeptide is conditionally essentialunder specific environmental or growth conditions. In other aspects, theconditionally essential polypeptide confers antibiotic resistance or isanother selection marker. In still other aspects, the recombinant cellis selected from the group consisting of a prokaryotic cell, aeukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion,a virosome, a virus-like particle, a plant cell, an animal cell, aninsect cell and a mammalian cell.

In certain exemplary embodiments, a recombinant cell including one ormore toxins or toxic substances that are inactive when a nonstandardamino acid is present in the one or more toxins or toxic substances at aparticular position is provided. The one or more toxins or toxicsubstances are activated when the nonstandard amino acid is not presentat the particular position, and the activated toxins or toxic substanceskill the recombinant cell or prevent or reduce proliferation of therecombinant cell. In certain aspects, one or both of folding andfunction of the one or more toxins or toxic substances are disruptedwhen the one or more toxins or toxic substances includes the nonstandardamino acid at the particular position. In certain aspects, the one ormore toxins or toxic substances are selected from the group consistingof one or any combination of barnase, Ccdb, Hok, Fst, ParE, MazF, Kid,ToxN, RelE, Doc, HipA and Mvpt. In certain aspects, the recombinant cellhas multiple copies of a nucleic acid sequence encoding the one or moretoxins or toxic substances. In yet other aspects, the cell is selectedfrom the group consisting of prokaryotic cell, a eukaryotic cell, ayeast cell, a bacterium, an archaeal cell, a virion, a virosome, avirus-like particle, a plant cell, an animal cell, an insect cell and amammalian cell.

In certain exemplary embodiments, a recombinant cell including anessential polypeptide producing or conferring susceptibility to one ormore toxins or toxic substances that are inactive when a nonstandardamino acid is present in the essential polypeptide at a particularposition is provided. The essential polypeptide is activated when thenonstandard amino acid is not present at the particular position, andthe activated essential polypeptide confers susceptibility to the one ormore toxins or toxic substances, which kills the recombinant cell orprevents or reduces proliferation of the recombinant cell. In certainaspects, one or both of folding and function of the essentialpolypeptide are disrupted when the essential polypeptide includes anonstandard amino acid at the particular position. In other aspects, theessential polypeptide produces or confers susceptibility to one or moretoxins or toxic substances under specific environmental or growthconditions. In certain aspects, the essential polypeptide is selectedfrom the group consisting of one or any combination of sacB tdk, galK,thyA, tolC, tetA, rpsL, and herpes simplex virus thymidine kinase. Inyet other aspects, multiple copies of a nucleic acid sequence encodingthe essential polypeptide are present in the recombinant cell. In yetother aspects, the cell is selected from the group consisting ofprokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, anarchaeal cell, a virion, a virosome, a virus-like particle, a plantcell, an animal cell, an insect cell and a mammalian cell.

In certain exemplary embodiments, a recombinant cell is providedincluding an essential polypeptide encoded by an essential nucleic acidsequence, said essential polypeptide having a nonstandard amino acidsubstitution, wherein the absence of the nonstandard amino acidsubstitution disrupts one or any combination of translation, folding andfunction of the essential polypeptide. The recombinant cell alsoincludes one or more toxins or toxic substances that are inactive when anonstandard amino acid is present in the one or more toxins or toxicsubstances at a particular position, wherein the one or more toxins ortoxic substances are activated when the nonstandard amino acid is notpresent at its particular position, and wherein the activated toxins ortoxic substances kill the recombinant cell or prevent or reduceproliferation of the recombinant cell. In certain aspects, the cell diesor experiences reduced or no proliferation if one or more oftranslation, folding or function is disrupted. In yet other aspects, thecell is selected from the group consisting of prokaryotic cell, aeukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion,a virosome, a virus-like particle, a plant cell, an animal cell, aninsect cell and a mammalian cell.

In certain exemplary embodiments, a recombinant cell is providedincluding an essential polypeptide encoded by an essential nucleic acidsequence, said essential polypeptide having a nonstandard amino acidsubstitution, wherein the absence of the nonstandard amino acidsubstitution disrupts one or any combination of translation, folding andfunction of the essential polypeptide. The recombinant cell alsoincludes an essential polypeptide producing or conferring susceptibilityto one or more toxins or toxic substances that are inactive when anonstandard amino acid is present in the essential polypeptide at aparticular position, wherein the essential polypeptide is activated whenthe nonstandard amino acid is not present at the particular position,and wherein the activated essential polypeptide confers susceptibilityto the one or more toxins or toxic substances, which kills therecombinant cell or prevents or reduces proliferation of the recombinantcell. In yet other aspects, the cell is selected from the groupconsisting of prokaryotic cell, a eukaryotic cell, a yeast cell, abacterium, an archaeal cell, a virion, a virosome, a virus-likeparticle, a plant cell, an animal cell, an insect cell and a mammaliancell.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1 schematically depicts positive selection for persistentdependence on NSAA incorporation for genetically modified organismviability. (Top panel): Wild-type core of essential protein (grey) witha natural amino acid to be reassigned (orange) and wild-type neighboringamino acid residues (green). (Middle panel): Natural amino acid isreassigned to an NSAA by genomic recoding while the neighboring residuesare redesigned to other natural amino acids that exclusively accommodatethe NSAA. (Bottom panel): Suppressor mutations to recoded gene or tRNAsresult in inviable cells due to misfolding of the essential protein whenthe suppressor amino acid is too small, leaving a cavity in the proteincore (lower left), or has the incorrect geometry, causing stericclashing with the core backbone or side-chains (lower right). Likewise,suppressors that introduce polar or charged amino acids into thehydrophobic core are likely to result in misfolded and/ornon-functioning protein, blocking cell viability.

FIG. 2 schematically depicts a synthetic auxotrophy approach accordingto certain embodiments. Panel A shows a toxin that is inactive in thepresence of the intended NSAA (+ symbols) at selected positions.Incorporation of any standard amino acid (− symbols) at the selectedposition due to suppression or mutagenesis leads to death of theorganism. Panel B shows an essential gene that requires the intendedNSAA to function. Withdrawal of the intended NSAA leads to death of theorganism.

FIG. 3 depicts a green fluorescent protein 1-UAG (stop codon) assay inwhich the specificities of the L-4,4′-Biphenylalanine (bipA) andL-3-(2-Naphthyl)alanine (napA) aminoacyl tRNA synthetases for theircognate NSAAs were assayed.

FIG. 4 graphically depicts optimization of L-4,4′-Biphenylalanine (bipA)and arabinose (ara) concentrations for the expression of GFP(fluorescence, induced by anhydrotetracycline (aTc)).

FIG. 5 schematically depicts adk strain results. Examples of escape(i.e., mutations conferring the ability to grow in the absence of bipA)and read-through (i.e., the strain is not fully dependent on bipAincorporation for survival) are indicated. SCAB refers to LB-Lennoxmedia supplemented with sodium dodecyl sulphate (SDS), chloramphenicol(Cm), arabinose (ara), and bipA. SCA refers to LB-Lennox mediasupplemented with SDS, Cm, and arabinose. SC refers to LB-Lennox mediasupplemented with SDS and Cm.

FIG. 6 graphically depicts the results of a growth assay of E. coliexpressing a recoded adenylate kinase (adk). This redesigned adk cloneexhibited dependence on bipA for survival, which was eventually overcomeby mutational escape.

FIG. 7 graphically depicts the results of a growth assay of E. coliexpressing a recoded alanyl-tRNA synthetase (alaS). This redesigned alaSclone exhibited dependence on bipA for survival, which was readilyovercome by mutational escape.

FIG. 8 graphically depicts the results of a growth assay of E. coliexpressing a recoded DNA polymerase III subunit delta (holB). Thisredesigned holB clone exhibited extremely impaired growth and lack ofdependence on bipA for survival.

FIG. 9 graphically depicts the results of a growth assay of E. coliexpressing a recoded methionyl-tRNA synthetase (metG). This redesignedmetG clone exhibited dependence on translation for survival, butincorporation of natural amino acids (in the SCA condition) appeared torescue fitness.

FIG. 10 graphically depicts the results of a growth assay of E. coliexpressing a recoded phosphoglycerate kinase (pgk). This redesigned pgkclone exhibited dependence on bipA for survival, which was eventuallyovercome by mutational escape.

FIG. 11 graphically compares growth assays of E. coli expressingphosphoglycerate kinase (pgk) (top panel) and alanyl-tRNA synthetasealaS (bottom panel). Without intending to be bound by scientific theory,lack of increased escape rate in the presence of arabinose (SCAcondition) suggests an alternative escape mechanism for pgk.

FIG. 12 graphically depicts the results of a growth assay of E. coliexpressing a recoded tyrosyl-tRNA synthetase (tyrS). This redesignedtyrS clone exhibited dependence on bipA for survival. No escape wasobserved after approximately 24 hours.

FIG. 13 graphically depicts growth assays of the top strains in thepresence of bipA. Most clones exhibited normal growth in the presence ofbipA, although pgk (green) and metG (orange) clones exhibited reducedfitness.

FIG. 14 depicts the range of escape frequencies of the top strains. ()=mutS+ interpolated by mutS−/100.

FIG. 15 depicts NSAAs suitable for use according to certain exemplaryembodiments of the invention (Liu and Schultz (2010) Ann. Rev. Biochem.79:413).

FIG. 16 depicts a table summarizing NSAAs suitable for use according tocertain exemplary embodiments of the invention. Id.

FIGS. 17A-17B depict NSAAs suitable for use according to certainexemplary embodiments of the invention (Kim et al. (2013) Curr. Opin.Chem. Biol. 17:412).

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

The subject application provides novel recombinant cells and organismspersistently expressing nonstandard amino acids (NSAAs) and methods ofmaking novel recombinant cells and organisms persistently expressingNSAAs.

As used herein, the term “amino acid” includes organic compoundscontaining both a basic amino group and an acidic carboxyl group.Included within this term are natural amino acids (e.g., L-amino acids),modified and unusual amino acids (e.g., D-amino acids and □-aminoacids), as well as amino acids which are known to occur biologically infree or combined form but usually do not occur in proteins.

As used herein, the term “NSAA” refers to an unmodified amino acid thatis not one of the 20 L-amino acids that typically naturally occur inproteins on Earth and includes alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, serine,threonine, tyrosine, tryptophan, proline and valine. NSAAs also refer tonatural amino acids that are not used by all organisms (e.g.L-pyrrolysine (B. Hao et al., A new uag-encoded residue in the structureof a methanogen methyltransferase. Science. 296:1462) andL-selenocysteine (S. Osawa et al., Recent evidence for evolution of thegenetic code. Microbiol. Mol. Biol. Rev. 56:229)). NSAAs are also knownin the art as unnatural amino acids (UAAs) and non-canonical amino acids(NCAAs).

NSAAs include, but are not limited to, p-Acetylphenylalanine,m-Acetylphenylalanine, O-allyltyrosine, Phenylselenocysteine,p-Propargyloxyphenylalanine, p-Azidophenylalanine,p-Boronophenylalanine, O-methyltyrosine, p-Aminophenylalanine,p-Cyanophenylalanine, m-Cyanophenylalanine, p-Fluorophenylalanine,p-Iodophenylalanine, p-Bromophenylalanine, p-Nitrophenylalanine, L-DOPA,3-Aminotyrosine, 3-Iodotyrosine, p-Isopropylphenylalanine,3-(2-Naphthyl)alanine, biphenylalanine, homoglutamine, D-tyrosine,p-Hydroxyphenyllactic acid, 2-Aminocaprylic acid, bipyridylalanine,HQ-alanine, p-Benzoylphenylalanine, o-Nitrobenzylcysteine,o-Nitrobenzylserine, 4,5-Dimethoxy-2-Nitrobenzylserine,o-Nitrobenzyllysine, o-Nitrobenzyltyrosine, 2-Nitrophenylalanine,dansylalanine, p-Carboxymethylphenylalanine, 3-Nitrotyrosine,sulfotyrosine, acetyllysine, methylhistidine, 2-Aminononanoic acid,2-Aminodecanoic acid, pyrrolysine, Cbz-lysine, Boc-lysine,allyloxycarbonyllysine, arginosuccinic acid, citrulline, cysteinesulfinic acid, 3,4-dihydroxyphenylalanine, homocysteine, homoserine,ornithine, 3-monoiodotyrosine, 3,5-diiodotryosine,3,5,5,-triiodothyronine, and 3,3′,5,5′-tetraiodothyronine. Modified orunusual amino acids include D-amino acids, hydroxylysine,4-hydroxyproline, N-Cbz-protected amino acids, 2,4-diaminobutyric acid,homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine,phenylglycine, □-phenylproline, tert-leucine, 4-aminocyclohexylalanine,N-methyl-norleucine, 3,4-dehydroproline, N,N-dimethylaminoglycine,N-methylaminoglycine, 4-aminopiperidine-4-carboxylic acid,6-aminocaproic acid, trans-4-(aminomethyl)-cyclohexanecarboxylic acid,2-, 3-, and 4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylicacid, 1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoicacid, any of the compounds disclose at FIG. 15, FIGS. 16A-16B, FIG. 17,and the like. NSAAs also include amino acids that are functionalized,e.g., alkyne-functionalized, azide-functionalized,ketone-functionalized, aminooxy-functionalized and the like. For reviewsof NSAAs and lists of NSAAs suitable for use in certain embodiments ofthe subject invention, see Liu and Schultz (2010) Ann. Rev. Biochem.79:413, and Kim et al. (2013) Curr. Opin. Chem. Biol. 17:412, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

In certain aspects, an NSAA of the subject invention has a correspondingaminoacyl tRNA synthetase (aaRS)/tRNA pair. In certain aspects, theaminoacyl tRNA synthetase/tRNA pair is orthogonal to those in agenetically modified organism such as, e.g., a prokaryotic cell, abacterium (e.g., E. coli), a eukaryotic cell, a yeast, a plant cell, aninsect cell, a mammalian cell, a virus, etc. In certain aspects, an NSAAof the subject invention is non-toxic when expressed in a geneticallymodified organism such as, e.g., a prokaryotic cell, a bacterium (e.g.,E. coli), a eukaryotic cell, a yeast, a plant cell, an insect cell, amammalian cell, a virus, etc. In certain aspects, an NSAA of the subjectinvention is not or does not resemble a natural product present in acell or organism. In certain aspects, an NSAA of the subject inventionis hydrophobic, hydrophilic, polar, positively charged, or negativelycharged. In other aspects, an NSAA of the subject invention iscommercially available (such as, e.g., bipA and L-2-Naphthylalanine(napA)) or synthesized according to published protocols.

As used herein, the term “peptide” includes compounds that consist oftwo or more amino acids that are linked by means of a peptide bond.Peptides may have a molecular weight of less than 10,000 Daltons, lessthan 5,000 Daltons, or less than 2,500 Daltons. The term “peptide” alsoincludes compounds containing both peptide and non-peptide components,such as pseudopeptide or peptidomimetic residues or other non-amino acidcomponents. Such compounds containing both peptide and non-peptidecomponents may also be referred to as a “peptide analog.”

As used herein, the terms “polypeptide” and “protein” include compoundsthat consist of amino acids arranged in a linear chain and joinedtogether by peptide bonds between the carboxyl and amino groups ofadjacent amino acid residues.

In certain exemplary embodiments, a recombinant cell or recombinantorganism of the subject invention comprises an “essential nucleic acidsequence” or “essential gene.” In certain aspects, expression of theessential nucleic acid sequence or essential gene produces an “essentialpolypeptide” or “essential protein.” In certain aspects, alternativenucleic acid sequences can encode for the same essential polypeptide. Ifone or more of translation, proper folding or proper functioning of theessential polypeptide encoded by the essential nucleic acid sequence oressential gene is disrupted, the recombinant cell or recombinantorganism dies or experiences reduced or no proliferation. Essentialnucleic acid sequences, essential genes, essential polypeptides oressential proteins can be involved with cell growth, cell division,housekeeping, cell death, and the like. Essential nucleic acidsequences, essential genes, essential polypeptides or essential proteinscan be conditionally essential (e.g., the ability to survive at highsalt concentrations, high heat conditions, during drought, etc.).Non-limiting, exemplary essential nucleic acid sequences and essentialgenes include, but are not limited to, tyrS, alaS, metS, metG, pgk, adk,holB and the like. Non-limiting, exemplary essential polypeptides andessential proteins include, but are not limited to, TyrS, AlaS, MetS,MetG, Pgk, Adk, HolB and the like. Using the subject disclosure as aguide, one of ordinary skill in the art could readily select additionalsuitable essential nucleic acid sequences and essential genes for usewith the recombinant cells, recombinant organisms and methods describedherein.

In certain exemplary embodiments, a recombinant cell or recombinantorganism of the subject invention comprises one or more toxins or toxicsubstances that kill the cell or organism or prevent or reduce itsproliferation. Non-limiting, exemplary toxins and toxic substancesinclude, but are not limited to, Ccdb, Hok, Fst, ParE, MazF, Kid, ToxN,RelE, Doc, HipA, Mvpt, SacB Tdk, GalK, ThyA, TolC, TetA, RpsL, barnase,and herpes simplex virus thymidine kinase. Using the subject disclosureas a guide, one of ordinary skill in the art could readily selectadditional suitable toxins and toxic substances for use with therecombinant cells, recombinant organisms and methods described herein.

In certain exemplary embodiments, a genomically recoded organismcomprising: a first essential polypeptide encoded by a first essentialgene, said first essential polypeptide having a first nonstandard aminoacid substitution, wherein the absence of the first nonstandard aminoacid substitution disrupts one or both of folding and function of thefirst essential polypeptide; and a second essential gene encoding apremature stop codon, wherein the presence of a second nonstandard aminosuppresses the premature stop codon and allows translation of a secondessential polypeptide is provided. In certain aspects, the organismcomprises two or more stop codons. In certain aspects, the organismfurther comprises one or more standard amino acid substitutions in thefirst essential polypeptide to accommodate the nonstandard amino acidsubstitution and maintain one or both of proper folding and properfunction of the first essential polypeptide. In certain aspects, thenonstandard amino acids are one or both of L-4,4′-Biphenylalanine andL-2-Naphthylalanine. In certain aspects, the first essential polypeptideis the same or is different than the second essential polypeptide. Incertain aspects, the nonstandard amino acid in the first essentialpolypeptide is the same or different than the nonstandard amino acid inthe second essential polypeptide. In certain aspects, the organismexpresses a plurality of nonstandard amino acids. In other aspects, oneor both of the first and second essential genes are selected from thegroup consisting of one or any combination of tyrS, alaS, pgk, metS,metG, adk, and holB. In certain aspects, the organism is weakened ordies if folding of the first essential polypeptide is disrupted, if afunction of the first essential polypeptide is disrupted, or iftranslation of the second essential polypeptide is terminated at thepremature stop codon. In still other aspects, the recombinant cell isselected from the group consisting of a prokaryotic cell, a eukaryoticcell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome,a virus-like particle, a plant cell, an animal cell, an insect cell anda mammalian cell.

In certain exemplary embodiments, a genomically recoded organismcomprising: 1) a first essential polypeptide encoded by an essentialgene, said first essential polypeptide having a first nonstandard aminoacid substitution, wherein the absence of the first nonstandard aminoacid substitution disrupts one or both of folding and function of thefirst essential polypeptide; 2) an essential gene encoding a prematurestop codon, wherein the presence of a second nonstandard aminosuppresses the premature stop codon and allows translation of a secondessential polypeptide; and 3) a toxin that is inactive when anonstandard amino acid is present in the toxin at a particular position,wherein the toxin is activated when the nonstandard amino acid is notpresent at the particular position, and wherein the activated toxinkills the organism. In certain aspects, the organism is weakened or diesif folding of the first essential polypeptide is disrupted, if afunction of the first essential polypeptide is disrupted, or iftranslation of the second polypeptide is terminated at the prematurestop codon. In still other aspects, the recombinant cell is selectedfrom the group consisting of a prokaryotic cell, a eukaryotic cell, ayeast cell, a bacterium, an archaeal cell, a virion, a virosome, avirus-like particle, a plant cell, an animal cell, an insect cell and amammalian cell.

The term “nucleoside,” as used herein, includes the natural nucleosides,including 2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kombergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.,described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980);Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990), or the like,with the proviso that they are capable of specific hybridization. Suchanalogs include synthetic nucleosides designed to enhance bindingproperties, reduce complexity, increase specificity, and the like.Polynucleotides comprising analogs with enhanced hybridization ornuclease resistance properties are described in Uhlman and Peyman (citedabove); Crooke et al., Exp. Opin. Ther. Patents, 6: 855-870 (1996);Mesmaeker et al., Current Opinion in Structural Biology, 5:343-355(1995); and the like. Exemplary types of polynucleotides that arecapable of enhancing duplex stability include oligonucleotidephosphoramidates (referred to herein as “amidates”), peptide nucleicacids (referred to herein as “PNAs”), oligo-2′-O-alkylribonucleotides,polynucleotides containing C-5 propynylpyrimidines, locked nucleic acids(LNAs), and like compounds. Such oligonucleotides are either availablecommercially or may be synthesized using methods described in theliterature.

“Oligonucleotide” or “polynucleotide,” which are used synonymously,means a linear polymer of natural or modified nucleosidic monomerslinked by phosphodiester bonds or analogs thereof. The term“oligonucleotide” usually refers to a shorter polymer, e.g., comprisingfrom about 3 to about 100 monomers, and the term “polynucleotide”usually refers to longer polymers, e.g., comprising from about 100monomers to many thousands of monomers, e.g., 10,000 monomers, or more.Oligonucleotides comprising probes or primers usually have lengths inthe range of from 12 to 60 nucleotides, and more usually, from 18 to 40nucleotides. Oligonucleotides and polynucleotides may be natural orsynthetic. Oligonucleotides and polynucleotides includedeoxyribonucleosides, ribonucleosides, and non-natural analogs thereof,such as anomeric forms thereof, peptide nucleic acids (PNAs), and thelike, provided that they are capable of specifically binding to a targetgenome by way of a regular pattern of monomer-to-monomer interactions,such as Watson-Crick type of base pairing, base stacking, Hoogsteen orreverse Hoogsteen types of base pairing, or the like.

Usually nucleosidic monomers are linked by phosphodiester bonds.Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGCCTG,” it will be understood that the nucleotides are in 5′to 3′ order from left to right and that “A” denotes deoxyadenosine, “C”denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotesdeoxythymidine, and “U” denotes the ribonucleoside, uridine, unlessotherwise noted. Usually oligonucleotides comprise the four naturaldeoxynucleotides; however, they may also comprise ribonucleosides ornon-natural nucleotide analogs. It is clear to those skilled in the artwhen oligonucleotides having natural or non-natural nucleotides may beemployed in methods and processes described herein. For example, whereprocessing by an enzyme is called for, usually oligonucleotidesconsisting solely of natural nucleotides are required. Likewise, wherean enzyme has specific oligonucleotide or polynucleotide substraterequirements for activity, e.g., single stranded DNA, RNA/DNA duplex, orthe like, then selection of appropriate composition for theoligonucleotide or polynucleotide substrates is well within theknowledge of one of ordinary skill, especially with guidance fromtreatises, such as Sambrook et al., Molecular Cloning, Second Edition(Cold Spring Harbor Laboratory, New York, 1989), and like references.Oligonucleotides and polynucleotides may be single stranded or doublestranded.

Oligonucleotides and polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides. Examples of modified nucleotides include, but are notlimited to diaminopurine, S²T, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone.

Nucleic acid molecules may be isolated from natural sources or purchasedfrom commercial sources. Oligonucleotide sequences may also be preparedby any suitable method, e.g., standard phosphoramidite methods such asthose described by Beaucage and Carruthers ((1981) Tetrahedron Lett. 22:1859) or the triester method according to Matteucci et al. (1981) J. Am.Chem. Soc. 103:3185), or by other chemical methods using either acommercial automated oligonucleotide synthesizer or high-throughput,high-density array methods known in the art (see U.S. Pat. Nos.5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813,5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference inits entirety for all purposes). Pre-synthesized oligonucleotides mayalso be obtained commercially from a variety of vendors.

In certain exemplary embodiments, recombinant cells and/or recombinantorganisms are provided that express one or more NSAAs. As used herein,an “organism” includes, but is not limited to, a human, a non-humanprimate, a cow, a horse, a sheep, a goat, a pig, a dog, a cat, a rabbit,a mouse, a rat, a gerbil, a frog, a toad, a fish (e.g., Danio rerio), aworm e.g., a roundworm (e.g., C. elegans), a plant, any eukaryote, anytransgenic species and any single or multiple cells derived therefrom.An organism or cell further includes, but is not limited to, a yeast(e.g., S. cerevisiae) cell, a yeast tetrad, a yeast colony, a bacterium,a bacterial colony, an archaeon, any prokaryote, a virion, a virosome, avirus-like particle, a parasitic microbe, an infectious protein and thelike and/or cultures of any of these.

In certain aspects, one or more biological samples are provided from oneor more recombinant organisms or one or more recombinant cells. As usedherein, a “biological sample” may be a single cell or many cells. Abiological sample may comprise a single cell type or a combination oftwo or more cell types. A biological sample further includes acollection of cells that perform a similar function such as those found,for example, in a tissue. As used herein, a tissue includes, but is notlimited to, epithelial tissue (e.g., skin, the lining of glands, bowel,skin and organs such as the liver, lung, kidney), endothelium (e.g., thelining of blood and lymphatic vessels), mesothelium (e.g., the lining ofpleural, peritoneal and pericardial spaces), mesenchyme (e.g., cellsfilling the spaces between the organs, including fat, muscle, bone,cartilage and tendon cells), blood cells (e.g., red and white bloodcells), neurons, germ cells (e.g., spermatozoa, oocytes), amniotic fluidcells, placenta, stem cells and the like. A tissue sample includesmicroscopic samples as well as macroscopic samples. In certain aspects,a biological sample is peripheral blood. In other aspects, a biologicalsample is a fluid such as saliva, synovial fluid, or the like. In stillother aspects, a biological sample is from one or more cell cultures,tissue sections and/or biopsies.

Isolation, extraction or derivation of nucleic acid sequences may becarried out by any suitable method. Isolating nucleic acid sequencesfrom a biological sample generally includes treating a biological samplein such a manner that nucleic acid sequences present in the sample areextracted and made available for analysis. Any isolation method thatresults in extracted nucleic acid sequences may be used in the practiceof the present invention. It will be understood that the particularmethod used to extract nucleic acid sequences will depend on the natureof the source.

Methods of DNA extraction are well-known in the art. A classical DNAisolation protocol is based on extraction using organic solvents such asa mixture of phenol and chloroform, followed by precipitation withethanol (J. Sambrook et al., “Molecular Cloning: A Laboratory Manual,”1989, 2^(nd) Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.).Other methods include: salting out DNA extraction (P. Sunnucks et al.,Genetics, 1996, 144: 747-756; S. M. Aljanabi and I. Martinez, Nucl.Acids Res. 1997, 25: 4692-4693), trimethylammonium bromide salts DNAextraction (S. Gustincich et al., BioTechniques, 1991, 11: 298-302) andguanidinium thiocyanate DNA extraction (J. B. W. Hammond et al.,Biochemistry, 1996, 240: 298-300). A variety of kits are commerciallyavailable for extracting DNA from biological samples (e.g., BDBiosciences Clontech (Palo Alto, Calif.): Epicentre Technologies(Madison, Wis.); Gentra Systems, Inc. (Minneapolis, Minn.); MicroProbeCorp. (Bothell, Wash.); Organon Teknika (Durham, N.C.); and Qiagen Inc.(Valencia, Calif.)).

Methods of RNA extraction are also well known in the art (see, forexample, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual”1989, 2′ Ed., Cold Spring Harbour Laboratory Press: New York) andseveral kits for RNA extraction from bodily fluids are commerciallyavailable (e.g., Ambion, Inc. (Austin, Tex.); Amersham Biosciences(Piscataway, N.J.); BD Biosciences Clontech (Palo Alto, Calif.); BioRadLaboratories (Hercules, Calif.); Dynal Biotech Inc. (Lake Success,N.Y.); Epicentre Technologies (Madison, Wis.); Gentra Systems, Inc.(Minneapolis, Minn.); GIBCO BRL (Gaithersburg, Md.); Invitrogen LifeTechnologies (Carlsbad, Calif.); MicroProbe Corp. (Bothell, Wash.);Organon Teknika (Durham, N.C.); Promega, Inc. (Madison, Wis.); andQiagen Inc. (Valencia, Calif.)).

Certain embodiments of the subject invention are directed to a firstnucleic acid or polypeptide sequence having a certain sequence identityor percent homology to a second nucleic acid or polypeptide sequence,respectively.

Techniques for determining nucleic acid and amino acid “sequenceidentity” are known in the art. Typically, such techniques includedetermining the nucleotide sequence of genomic DNA, mRNA or cDNA madefrom an mRNA for a gene and/or determining the amino acid sequence thatit encodes, and comparing one or both of these sequences to a secondnucleotide or amino acid sequence, as appropriate. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. Anexemplary implementation of this algorithm to determine percent identityof a sequence is provided by the Genetics Computer Group (Madison, Wis.)in the “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.).

One method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages, the Smith-Waterman algorithm canbe employed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at theNCBI/NLM web site.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions that form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. Two DNAsequences, or two polypeptide sequences are “substantially homologous”to each other when the sequences exhibit at least about 80%-85%, atleast about 85%-90%, at least about 90%-95%, or at least about 95%-98%,or about 95%, about 96%, about 97%, about 98%, or about 99% sequenceidentity over a defined length of the molecules, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.; Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supra).Such assays can be conducted using varying degrees of selectivity, forexample, using conditions varying from low to high stringency. Ifconditions of low stringency are employed, the absence of non-specificbinding can be assessed using a secondary probe that lacks even apartial degree of sequence identity (for example, a probe having lessthan about 30% sequence identity with the target molecule), such that,in the absence of non-specific binding events, the secondary probe willnot hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” conditionstypically hybridizes under conditions that allow detection of a targetnucleic acid sequence of at least about 10-14 nucleotides in lengthhaving at least approximately 70% sequence identity with the sequence ofthe selected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization, supra).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook et al., supra).

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% identical to each othertypically remain hybridized to each other. In one aspect, the conditionsare such that sequences at least about 70%, at least about 80%, at leastabout 85% or 90% or more identical to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A non-limitingexample of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50° C., at 55° C., or at 60° C. or65° C.

In certain exemplary embodiments, methods for amplifying nucleic acidsequences are provided. Exemplary methods for amplifying nucleic acidsinclude the polymerase chain reaction (PCR) (see, e.g., Mullis et al.(1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary etal. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see,e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364), self-sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.U.S.A. 87:1874), transcriptional amplification system (Kwoh et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardiet al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000)J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem.277:7790), the amplification methods described in U.S. Pat. Nos.6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199,isothermal amplification (e.g., rolling circle amplification (RCA),hyperbranched rolling circle amplification (HRCA), strand displacementamplification (SDA), helicase-dependent amplification (HDA), PWGA) orany other nucleic acid amplification method using techniques well knownto those of skill in the art.

“Polymerase chain reaction,” or “PCR,” refers to a reaction for the invitro amplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g., exemplified by the references:McPherson et al., editors, PCR: A Practical Approach and PCR2: APractical Approach (IRL Press, Oxford, 1991 and 1995, respectively). Forexample, in a conventional PCR using Taq DNA polymerase, a doublestranded target nucleic acid may be denatured at a temperature greaterthan 90° C., primers annealed at a temperature in the range 50-75° C.,and primers extended at a temperature in the range 72-78° C.

The term “PCR” encompasses derivative forms of the reaction, includingbut not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, assembly PCR and the like. Reaction volumes range froma few hundred nanoliters, e.g., 200 nL, to a few hundred microliters,e.g., 200 microliters. “Reverse transcription PCR,” or “RT-PCR,” means aPCR that is preceded by a reverse transcription reaction that converts atarget RNA to a complementary single stranded DNA, which is thenamplified, e.g., Tecott et al., U.S. Pat. No. 5,168,038. “Real-time PCR”means a PCR for which the amount of reaction product, i.e., amplicon, ismonitored as the reaction proceeds. There are many forms of real-timePCR that differ mainly in the detection chemistries used for monitoringthe reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015(“Taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627(intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecularbeacons). Detection chemistries for real-time PCR are reviewed in Mackayet al., Nucleic Acids Research, 30:1292-1305 (2002). “Nested PCR” meansa two-stage PCR wherein the amplicon of a first PCR becomes the samplefor a second PCR using a new set of primers, at least one of which bindsto an interior location of the first amplicon. As used herein, “initialprimers” in reference to a nested amplification reaction mean theprimers used to generate a first amplicon, and “secondary primers” meanthe one or more primers used to generate a second, or nested, amplicon.“Multiplexed PCR” means a PCR wherein multiple target sequences (or asingle target sequence and one or more reference sequences) aresimultaneously carried out in the same reaction mixture, e.g. Bernard etal. (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR).Usually, distinct sets of primers are employed for each sequence beingamplified. “Quantitative PCR” means a PCR designed to measure theabundance of one or more specific target sequences in a sample orspecimen. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following references:Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al.,Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al.,Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020(1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446(1989); and the like.

In certain exemplary embodiments, methods of determining the sequenceidentities of nucleic acid sequences are provided. Determination of thesequence of a nucleic acid sequence of interest (e.g., immune cellnucleic acid sequences) can be performed using variety of sequencingmethods known in the art including, but not limited to, sequencing byhybridization (SBH), sequencing by ligation (SBL), quantitativeincremental fluorescent nucleotide addition sequencing (QIFNAS),stepwise ligation and cleavage, fluorescence resonance energy transfer(FRET), molecular beacons, TaqMan reporter probe digestion,pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads(U.S. Pat. No. 7,425,431), wobble sequencing (PCT/US05/27695), multiplexsequencing (U.S. Ser. No. 12/027,039, filed Feb. 6, 2008; Porreca et al(2007) Nat. Methods 4:931), polymerized colony (POLONY) sequencing (U.S.Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425);nanogrid rolling circle sequencing (ROLONY) (U.S. Ser. No. 12/120,541,filed May 14, 2008), allele-specific oligo ligation assays (e.g., oligoligation assay (OLA), single template molecule OLA using a ligatedlinear probe and a rolling circle amplification (RCA) readout, ligatedpadlock probes, and/or single template molecule OLA using a ligatedcircular padlock probe and a rolling circle amplification (RCA) readout)and the like. High-throughput sequencing methods, e.g., on cyclic arraysequencing using platforms such as Roche 454, Illumina Solexa,ABI-SOLiD, ION Torrents, Complete Genomics, Pacific Bioscience, Helicos,Polonator platforms (Worldwide Web Site: Polonator.org), and the like,can also be utilized. High-throughput sequencing methods are describedin U.S. Ser. No. 61/162,913, filed Mar. 24, 2009. A variety oflight-based sequencing technologies are known in the art (Landegren etal. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100;and Shi (2001) Clin. Chem. 47:164-172).

Embodiments of the invention include the use of computer software tocomputationally design and/or analyze nucleic acid sequences and/orpolypeptides described herein. Such software may be used in conjunctionwith individuals performing design and/or analysis by hand or in asemi-automated fashion or combined with an automated system. In at leastsome embodiments, the gene/oligonucleotide design/analysis software isimplemented in a program written in the JAVA programming language. Theprogram may be compiled into an executable that may then be run from acommand prompt in the WINDOWS XP operating system. The invention issimilarly not limited to implementation using a specific software code,programming language, operating system environment or hardware platform.In certain aspects, protein design may be performed using Rosettasoftware. (See, e.g., U.S. Pat. No. 8,340,951, incorporated herein byreference in its entirety for all purposes.)

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures andaccompanying claims.

Example I Positive and Negative Selection Strategies

According to certain exemplary embodiments, genetically modifiedorganisms were provided and maintained using a positive selection forNSAA incorporation. NSAA-dependent genetically modified organisms wereengineered by reassigning the UAG codon to incorporate an NSAA at eitherN-terminal or solvent-exposed positions in essential genes. Since thesepositions are subject to suppression by standard amino acids, thisapproach has been extended to engineer essential genes that require theNSAA for folding and/or function in addition to translation. Acomputational second-site suppressor strategy (T. Kortemme et al. (2004)Computational redesign of protein-protein interaction specificity. Nat.Struct. Mol. Biol. 11:371) was employed to redesign the hydrophobiccores of essential genes in this fashion. This approach mutated corepositions in essential genes to an NSAA, and then redesigned theneighboring amino acid residues to exclusively accommodate theintroduced NSAA. Suppression of the NSAA then disrupted folding and/orfunction of the essential protein. This strategy was applied to the corepositions of all essential genes in E. coli with available X-raystructures (141 genes) using L-4,4′-biphenylalanine as the NSAA. NSAAdependence was assayed in strains with redesigned essential genesincluding tyrS, alaS, holB, mete, pgk and adk.

In certain exemplary embodiments, genetically modified organisms areprovided and maintained using a negative selection for NSAAincorporation. Mutations to endogenous or engineered tRNAs can producesuppressors that incorporate natural amino acids at recoded positions,compromising safety features that rely on recoding. In order to selectagainst suppressors arising, natural toxins are engineered with foldingand/or function that is disrupted only when an NSAA is incorporated atrecoded positions. Incorporation of a natural amino acid at a recodedposition then activates the toxin and kills the cell. Since certainpositions may be amenable to excluding some but not all natural aminoacids, this strategy is applied across multiple positions and toxins,including barnase, ccdb, hok, fst, parE, mazF, kid, toxN, relE, doc,hipA and mvpt. Multiple copies may be included to mitigate loss of thetoxin genes via mutational inactivation, deletion, or the like.

Example II Protein Design Using Second-Site Strategy

Plasmids were assembled containing the aaRS and tRNA for bipA and napA(2 copies each, one constitutive, one under arabinose-inducibletranscriptional control). Incorporation and specificity were assayedusing a GFP with 1 UAG codon (FIG. 3). The assay indicated high activityfor the cognate NSAA and high specificity (low misincorporation ofstandard amino acids).

Rosetta was used to apply the second-site suppressor strategy toreengineer essential proteins to exclusively accommodate bipA and napAat certain positions. A set of 129 high-resolution (2.8 Å) X-raystructures of essential E. coli proteins was compiled. The solventaccessible surface area (SASA) of all residues in all proteins wascomputed, and 13,564 “buried” residues were identified. All buriedpositions were redesigned to incorporate an NSAA. Neighbors wereredesigned to accommodate an NSAA. Best candidates were identified byselecting redesigned protein cores that were tightly packed around theNSAA, which were thus predicted to be destabilized in the presence ofany standard amino acid. It was determined that the target site was notproximal to residues critical for catalysis, substrate binding, orallosteric transduction. It was determined that the same product or asimilar product was not found in nature. Best candidates were refinedwith another round of design with manually selected degrees of freedom.

Design protocols resulted in six essential polypeptides that werepursued further experimentally. BipA simulations produced more promisingdesigns. Without intending to be bound by scientific theory, this waslikely due to the presence of more conformations because of the extrarotatable bond, and/or due to difficulties in suppression due to largercavity formed. The six bipA target systems were as follows: 1) adenylatekinase (adk) (an essential enzyme required for the biosynthesis ofpurine ribonucleotides); 2) alanyl-tRNA synthetase (alaS); 3) DNApolymerase III subunit delta (holB); 4) methionyl-tRNA synthetase(metG); 5) phosphoglycerate kinase (pgk) (produces 3-Phosphoglycericacid, a metabolic intermediate in glycolysis); and 6) tyrosyl-tRNAsynthetase (tyrS).

Targets were scattered across the genome, so that multiple redesignsmade gene transfer difficult. Out of 4.6 MB, the genomic position ofeach of the six essential polypeptides was as follows: adk:496,399->497,043; alaS: 2,817,403<-2,820,033; holB:1,154,985->1,155,989; metG: 2,192,322->2,194,355; pgk:3,069,481<-3,070,644; and tyrS: 1,713,972<-1,715,246. (Numbers representleft and right genome positions of the gene; arrows representorientation of the gene (-> is a gene transcribed on the + strand; <- isa gene transcribed on the − strand).)

Example III Two Methods for Designing Strains

Designed strains were constructed in two ways:

Fewer Compensatory Mutations (all Targets)

-   -   1. Encoded desired mutations on degenerate oligos    -   2. Introduced tolC cassette near target gene using lambda red        recombineering    -   3. Inactivated tolC using lambda red recombineering    -   4. Introduced oligos to mutate the essential gene and to        reactivate tolC using lambda red recombineering    -   5. Replica plated with/without bipA to identify bipA-dependent        clones    -   6. Phenotypic (bipA dependence) and genotypic (PCR and        sequencing) screening of recombinants

More Compensatory Mutations (adk, tyrS)

-   -   1. Encoded desired mutations in degenerate IDT gBlocks (500 bp        synthetic double-stranded DNA constructs)    -   2. Assembled into full genes using isothermal assembly and/or        PCR assembly    -   3. Inserted a copy of wild type essential gene at tolC locus by        using lambda red recombineering    -   4. Deleted essential gene locus with tolC by using lambda red        recombineering    -   5. Replaced tolC in wild type locus with redesigned essential        gene by using lambda red recombineering    -   6. Deleted redundant copy of wild type essential gene by using        lambda red recombineering to reintroduce tolC into tolC locus    -   7. Phenotypic (bipA dependence) and genotypic (PCR and        sequencing) screening of recombinants

Candidates showed different levels of dependence and different escapemechanisms (FIG. 5). Without intending to be bound by scientific theory,there were several reasons for growth in the absence of bipA. “Readthrough” or “bleed through,” in which near-cognate suppression bynatural tRNAs incorporating natural amino acids resulted in adequateexpression and function of the essential polypeptide. Escapesuppressors: mutations in the redesigned essential polypeptide relievingbipA dependence, mutations in natural tRNAs conferring the ability toincorporate natural amino acids at the UAG codon that are capable ofrescuing polypeptide function, or mutations in the bipA aaRS and/or tRNAallowing them to recognize natural tRNAs or natural amino acids.

“Ideal” compensatory second-site mutations should prevent both “bleedthrough” and “escape suppressors,” which is very challenging. Moststrains identified were mutator strains (mismatch repair is inactivated,leading to a 100-fold increase in mutation frequency). This increasedmutation load allowed detection of rare escape mechanisms, but escapefrequencies could be reduced approximately 100-fold by reintroducingmismatch repair. Synthetase and tRNA were on a 10-20 copy plasmid or insingle copy on the genome.

Example IV Genomically Recoded Strain Analysis

Adenylate Kinase (Adk)

Adenylate kinase (adk) having the following standard and nonstandardamino acid substitutions was generated: I4A, L6V, V103A, L178bipA,Y182V, T191I. Results of growth assays are depicted at FIG. 6.

Alanyl-tRNA Synthetase (alaS)

Alanyl-tRNA synthetase (alaS) having the following standard andnonstandard amino acid substitutions was generated: startUAG, F90A,F293A, L338bipA, M342A, L349P. Results of growth assays are depicted atFIG. 7.

DNA Polymerase III Subunit Delta (holB)

DNA polymerase III subunit delta (holB) having an A190bipA substitutionwas generated. Results of growth assays are depicted at FIG. 8. The onlysuccessful substitution in holB was UAG. Without intending to be boundby scientific theory, it is possible that recombination frequencies weretoo low, but it was more likely that the redesign was not compatiblewith holB folding and/or function

Methionyl-tRNA Synthetase (metG)

Methionyl-tRNA synthetase (metG) having the following standard andnonstandard amino acid substitutions was generated: M485A, F502G,L503bipA. Results of growth assays are depicted at FIG. 9.

Phosphoglycerate Kinase (Pgk)

Phosphoglycerate kinase (pgk) having the following standard andnonstandard amino acid substitutions was generated: V185A, I187A, I211G,L297bipA. Results of growth assays are depicted at FIG. 10.Phosphoglycerate kinase may have an escape mechanism that is unaffectedby bipA aaRS/tRNA expression, as evidenced by equivalent growth profilesin presence and absence of arabinose. See FIG. 11.

Tyrosyl-tRNA Synthetase (tyrS)

Tyrosyl-tRNA synthetase (tyrS) having the following standard andnonstandard amino acid substitutions was generated: F706A, W778F, T787V,F823G, L907bipA, V919A. Results of growth assays are depicted at FIG.12.

Most of the best candidates showed normal growth phenotype in thepresence of bipA (FIG. 13). Best candidates had a range of escapefrequencies (FIG. 14).

Example IV Additional Strategies

Escape mechanisms are determined using, e.g., NNN oligos andnext-generation sequencing methods. Best candidates are combined byconjugation, e.g., mutS+, bipA synthetase on genome, and/ormultiplicative effect from orthogonal escape routes.

Saturation mutagenesis is performed for all recoded sites and tRNAs toensure that there are no pathways to escape.

X-Ray Structures are Solved for Essential Proteins with NSAAs inRedesigned Cores

Environmental studies of safe genetically modified organisms areperformed using a variety of substrates including, but not limited to,soil, water, waste, compost, and the like.

Conjugation studies to assess escape of safe genetically modifiedorganisms due to horizontal gene transfer are performed.

Additional codons are reassigned to increase the genetic isolation ofgenetically modified organisms.

EQUIVALENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above example, but areencompassed by the claims. All publications, patents and patentapplications cited above are incorporated by reference herein in theirentirety for all purposes to the same extent as if each individualpublication or patent application were specifically indicated to be soincorporated by reference.

1.-17. (canceled)
 18. A genetically modified cell comprising: anessential polypeptide producing or conferring susceptibility to one ormore toxins or toxic substances that are inactive when a nonstandardamino acid is present in the essential polypeptide at a particularposition, wherein the essential polypeptide is activated when thenonstandard amino acid is not present at the particular position, andwherein the activated essential polypeptide confers susceptibility tothe one or more toxins or toxic substances, which kills the recombinantcell or prevents or reduces proliferation of the recombinant cell. 19.The genetically modified cell of claim 18, wherein one or both offolding and function of the essential polypeptide are disrupted when theessential polypeptide includes a nonstandard amino acid at theparticular position.
 20. The genetically modified cell of claim 18,wherein the essential polypeptide produces or confers susceptibility toone or more toxins or toxic substances under specific environmental orgrowth conditions.
 21. The genetically modified cell of claim 20, inwhich the essential polypeptide is selected from the group consisting ofone or any combination of SacB Tdk, GalK, ThyA, TolC, TetA, RpsL, andherpes simplex virus thymidine kinase.
 22. The genetically modified cellof claim 18, wherein multiple copies of a nucleic acid sequence encodingthe essential polypeptide are present in the recombinant cell.
 23. Thegenetically modified cell of claim 18, wherein the recombinant cell isselected from the group consisting of a prokaryotic cell, a eukaryoticcell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome,a virus-like particle, a plant cell, an animal cell, an insect cell anda mammalian cell.
 24. A The genetically modified cell of claim 18further comprising: one or more toxins or toxic substances that areinactive when a nonstandard amino acid is present in the one or moretoxins or toxic substances at a particular position, wherein the one ormore toxins or toxic substances are activated when the nonstandard aminoacid is not present at its particular position, and wherein theactivated toxins or toxic substances kill the recombinant cell orprevent or reduce proliferation of the recombinant cell.
 25. Thegenetically modified cell of claim 24, wherein the cell dies orexperiences reduced or no proliferation if one or more of translation,folding or function is disrupted.
 26. The genetically modified cell ofclaim 24, wherein the cell is selected from the group consisting of aprokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, anarchaeal cell, a virion, a virosome, a virus-like particle, a plantcell, an animal cell, an insect cell and a mammalian cell.
 27. Agenetically modified cell comprising: 1) an essential polypeptideencoded by an essential nucleic acid sequence, said essentialpolypeptide having a nonstandard amino acid substitution, wherein theabsence of the nonstandard amino acid substitution disrupts one or anycombination of translation, folding and function of the essentialpolypeptide; and 2) an essential polypeptide producing or conferringsusceptibility to one or more toxins or toxic substances that areinactive when a nonstandard amino acid is present in the essentialpolypeptide at a particular position, wherein the essential polypeptideis activated when the nonstandard amino acid is not present at theparticular position, and wherein the activated essential polypeptideconfers susceptibility to the one or more toxins or toxic substances,which kills the recombinant cell or prevents or reduces proliferation ofthe recombinant cell.
 28. The genetically modified cell of claim 27,wherein the cell dies or experiences reduced or no proliferation if oneor more of translation, folding or function is disrupted.
 29. Thegenetically modified cell of claim 27, wherein the cell is selected fromthe group consisting of a prokaryotic cell, a eukaryotic cell, a yeastcell, a bacterium, an archaeal cell, a virion, a virosome, a virus-likeparticle, a plant cell, an animal cell, an insect cell and a mammaliancell.
 30. A genomically recoded organism comprising: 1) a firstessential polypeptide encoded by a first essential gene, said firstessential polypeptide having a first nonstandard amino acidsubstitution, wherein the absence of the first nonstandard amino acidsubstitution disrupts one or both of folding and function of the firstessential polypeptide; and 2) a second essential gene encoding apremature stop codon, wherein the presence of a second nonstandard aminosuppresses the premature stop codon and allows translation of a secondessential polypeptide.
 31. The genomically recoded organism of claim 30,comprising two or more stop codons.
 32. The genomically recoded organismof claim 30, further comprising one or more standard amino acidsubstitutions in the first essential polypeptide to accommodate thenonstandard amino acid substitution and maintain one or both of properfolding and proper function of the first essential polypeptide.
 33. Thegenomically recoded organism of claim 30, wherein the nonstandard aminoacids are one or both of L-4,4′-Biphenylalanine and L-2-Naphthylalanine.34. The genomically recoded organism of claim 30, wherein the firstessential polypeptide is the same as the second essential polypeptide.35. The genomically recoded organism of claim 30, wherein the firstessential polypeptide is different than the second essentialpolypeptide.
 36. The genomically recoded organism of claim 30, whereinthe nonstandard amino acid in the first essential polypeptide is thesame as the nonstandard amino acid in the second essential polypeptide.37. The genomically recoded organism of claim 30, wherein thenonstandard amino acid in the first essential polypeptide is differentthan the nonstandard amino acid in the second essential polypeptide. 38.The genomically recoded organism of claim 30, wherein the organismexpresses a plurality of nonstandard amino acids.
 39. The genomicallyrecoded organism of claim 30, wherein one or both of the first andsecond essential genes are selected from the group consisting of one orany combination of tyrS, alaS, pgk, metS, metG, adk, and holB.
 40. Thegenomically recoded organism of claim 30, wherein the organism isweakened or dies if folding of the first essential polypeptide isdisrupted, if a function of the first essential polypeptide isdisrupted, or if translation of the second essential polypeptide isterminated at the premature stop codon.
 41. The genomically recodedorganism of claim 30, wherein the organism is selected from the groupconsisting of a yeast cell, a bacterium, a virion, a virosome, avirus-like particle, a plant cell, an insect cell, and a mammalian cell.42. The genomically recoded organism of claim 30 further comprising: atoxin that is inactive when a nonstandard amino acid is present in thetoxin at a particular position, wherein the toxin is activated when thenonstandard amino acid is not present at the particular position, andwherein the activated toxin kills the organism.
 43. The genomicallyrecoded organism of claim 42, wherein the organism is weakened or diesif folding of the first essential polypeptide is disrupted, if afunction of the first essential polypeptide is disrupted, or iftranslation of the second polypeptide is terminated at the prematurestop codon.
 44. The genomically recoded organism of claim 42, whereinthe organism is selected from the group consisting of a yeast cell, abacterium, a virion, a virosome, a virus-like particle, a plant cell, aninsect cell, and a mammalian cell.
 45. A method of making anNSAA-dependent genetically modified cell comprising: providing to thecell an essential polypeptide encoded by an essential nucleic acidsequence, said essential polypeptide having a nonstandard amino acidsubstitution at a particular position, wherein the absence of thenonstandard amino acid substitution at the particular position disruptsone or any combination of translation, folding and function of theessential polypeptide, and wherein the recombinant cell dies orexperiences reduced or no proliferation if one or more of translation,folding or function of the essential polypeptide is disrupted.
 46. Themethod of claim 45, wherein the essential polypeptide further comprisesone or more standard amino acid substitutions in the essentialpolypeptide to accommodate the nonstandard amino acid substitution andmaintain one or both of proper folding and proper function of theessential polypeptide.
 47. The method of claim 45, wherein the essentialpolypeptide has been computationally redesigned so that the essentialpolypeptide can no longer fold properly, function properly or both, whenthe essential polypeptide expresses a standard amino acid at theparticular position.
 48. The method of claim 47, wherein the redesign isperformed by random mutagenesis and selection.
 49. The method of claim47, wherein the redesign is performed by targeted mutagenesis andselection.
 50. The method of claim 47, wherein the redesign is performedcomputationally using Rosetta software.
 51. The method of claim 45,wherein the essential polypeptide is conditionally essential underspecific environmental or growth conditions.
 52. The method of claim 45,wherein the conditionally essential polypeptide confers antibioticresistance or is another selection marker.
 53. The method of claim 45,further comprising providing to the cell two or more essentialpolypeptides encoded by two or more essential nucleic acid sequences.54. The method of claim 45, wherein the cell expresses a plurality ofnonstandard amino acids.
 55. The method of claim 45, wherein theessential nucleic acid sequence is an essential gene or encodes anessential polypeptide that is essential for survival, growth, orproliferation.
 56. The method of claim 45, wherein the cell is selectedfrom the group consisting of prokaryotic cell, a eukaryotic cell, ayeast cell, a bacterium, an archaeal cell, a virion, a virosome, avirus-like particle, a plant cell, an animal cell, an insect cell and amammalian cell.
 57. The method of claim 45, further comprising providingto the cell one or more toxins or toxic substances that are inactivewhen a nonstandard amino acid is present in the one or more toxins ortoxic substances at a particular position, wherein the one or moretoxins or toxic substances are activated when the nonstandard amino acidis not present at its particular position, and wherein the activatedtoxins or toxic substances kill the recombinant cell or prevent orreduce proliferation of the recombinant cell.
 58. The method of claim57, wherein one or both of folding and function of the one or moretoxins or toxic substances are disrupted when the one or more toxins ortoxic substances includes the nonstandard amino acid at the particularposition.
 59. The method of claim 57, wherein the one or more toxins ortoxic substances are selected from the group consisting of one or anycombination of barnase, Ccdb, Hok, Fst, ParE, MazF, Kid, ToxN, RelE,Doc, HipA and Mvpt.
 60. The method of claim 57, wherein multiple copiesof a nucleic acid sequence encoding the one or more toxins or toxicsubstances are provided to the cell.
 61. The method of claim 57, whereinthe cell is selected from the group consisting of a prokaryotic cell, aeukaryotic cell, a yeast cell, a bacterium, an archaeal cell, a virion,a virosome, a virus-like particle, a plant cell, an animal cell, aninsect cell and a mammalian cell.
 62. A method of conferringsusceptibility to one or more toxins or toxic substance to a cellcomprising: providing to the cell an essential polypeptide producing orconferring susceptibility to one or more toxins or toxic substances thatare inactive when a nonstandard amino acid is present in the essentialpolypeptide at a particular position, wherein the essential polypeptideis activated when the nonstandard amino acid is not present at theparticular position, and wherein the activated essential polypeptideconfers susceptibility to the one or more toxins or toxic substances,which kills the recombinant cell or prevents or reduces proliferation ofthe recombinant cell.
 63. The method of claim 62, wherein one or both offolding and function of the essential polypeptide are disrupted when theessential polypeptide includes a nonstandard amino acid at theparticular position.
 64. The method of claim 62, wherein the essentialpolypeptide produces or confers susceptibility to one or more toxins ortoxic substances under specific environmental or growth conditions. 65.The method of claim 62, wherein the essential polypeptide is selectedfrom the group consisting of one or any combination of SacB Tdk, GalK,ThyA, TolC, TetA, RpsL, and herpes simplex virus thymidine kinase. 66.The method of claim 62, wherein multiple copies of a nucleic acidsequence encoding the essential polypeptide are provided to the cell.67. The method of claim 62, wherein the cell is selected from the groupconsisting of a prokaryotic cell, a eukaryotic cell, a yeast cell, abacterium, an archaeal cell, a virion, a virosome, a virus-likeparticle, a plant cell, an animal cell, an insect cell and a mammaliancell.
 68. The method of claim 62, further comprising providing to thecell one or more toxins or toxic substances that are inactive when anonstandard amino acid is present in the one or more toxins or toxicsubstances at a particular position, wherein the one or more toxins ortoxic substances are activated when the nonstandard amino acid is notpresent at its particular position, and wherein the activated toxins ortoxic substances kill the cell or prevent or reduce proliferation of thecell.
 69. The method of claim 68, wherein the cell dies or experiencesreduced or no proliferation if one or more of translation, folding orfunction is disrupted.
 70. The method of claim 68, wherein the cell isselected from the group consisting of a prokaryotic cell, a eukaryoticcell, a yeast cell, a bacterium, an archaeal cell, a virion, a virosome,a virus-like particle, a plant cell, an animal cell, an insect cell anda mammalian cell.
 71. A method of making an NSAA-dependent geneticallymodified cell that is susceptible to one or more toxins or toxicsubstance comprising providing to the cell 1) an essential polypeptideencoded by an essential nucleic acid sequence, said essentialpolypeptide having a nonstandard amino acid substitution, wherein theabsence of the nonstandard amino acid substitution disrupts one or anycombination of translation, folding and function of the essentialpolypeptide; and 2) an essential polypeptide producing or conferringsusceptibility to one or more toxins or toxic substances that areinactive when a nonstandard amino acid is present in the essentialpolypeptide at a particular position, wherein the essential polypeptideis activated when the nonstandard amino acid is not present at theparticular position, and wherein the activated essential polypeptideconfers susceptibility to the one or more toxins or toxic substances,which kills the recombinant cell or prevents or reduces proliferation ofthe recombinant cell.
 72. The method of claim 71, wherein the cell diesor experiences reduced or no proliferation if one or more oftranslation, folding or function is disrupted.
 73. The method of claim71, wherein the cell is selected from the group consisting of aprokaryotic cell, a eukaryotic cell, a yeast cell, a bacterium, anarchaeal cell, a virion, a virosome, a virus-like particle, a plantcell, an animal cell, an insect cell and a mammalian cell.